Certain P-450s are developmentally regulated. These include testosterone-16Alpha-hydroxylase, an adult male specific P-450, and testosterone-15Alpha-hydroxylase, an adult female specific P-450. These two enzymes increase during rat development and their increase is mediated through blood androgen levels and pulsatile levels of growth hormone. We have been analyzing two classes of P-450s: one class of P-450, including P-450f and P-450PB-1, have no known steroid hydroxylase activities. These P-450s increase during development in both males and females and reach maximal levels at rat maturity. This increase is independent of androgens. We cloned and sequenced cDNAs for these enzymes and found that they share 75% amino acid homology. The cDNA probes were used to show that these P-450s increase during development as a result of transcriptional activation of their respective genes. These P-450s are probably linked and are coordinately regulated. The cDNA of a P-450 that is induced by steroids was cloned and sequenced. This P-450, designated P-450PCN1 (PCN, pregnenolone-16Alpha-carbonitrile), was found to be absent in noninduced rats and induced by PCN and phenobarbital. Another P-450 cDNA was isolated based on its sequence homology with P-450PCN1 and designated P-450PCN2. P-450PCN2 has 90% amino acid sequence similarity with P-450PCN1. This homology is present in three distinct regions of 99% to 100% similarity surrounded by regions of 80 to 85% similarity. These data suggest that gene conversion events have played a role in evolution of this P-450 family. Interestingly, P-450PCN2 is a developmentally regulated P-450. Its level increases in young male and female rats but declines in female rats at puberty and continues to increase in male rats, reaching maximal levels at maturity. In contrast to P-450CN1, P-450PCN2 is not induced by steroids such as PCN and dexamethasone. Similar to P-450PCN1, P-450PCN2 is induced by phenobarbital. Induction and developmental studies suggest that both P-450s possess testosterone-6Betahydroxylase activity.